Which Constituents Determine the Antioxidant Activity and Cytotoxicity of Garlic? Role of Organosulfur Compounds and Phenolics

Garlic is a vegetable with numerous pro-health properties, showing high antioxidant capacity, and cytotoxicity for various malignant cells. The inhibition of cell proliferation by garlic is mainly attributed to the organosulfur compounds (OSCs), but it is far from obvious which constituents of garlic indeed participate in the antioxidant and cytotoxic action of garlic extracts. This study aimed to obtain insight into this question by examining the antioxidant activity and cytotoxicity of six OSCs and five phenolics present in garlic. Three common assays of antioxidant activity were employed (ABTS● decolorization, DPPH● decolorization, and FRAP). Cytotoxicity of both classes of compounds to PEO1 and SKOV-3 ovarian cancer cells, and MRC-5 fibroblasts was compared. Negligible antioxidant activities of the studied OSCs (alliin, allicin, S-allyl-D-cysteine, allyl sulfide, diallyl disulfide, and diallyl trisulfide) were observed, excluding the possibility of any significant contribution of these compounds to the total antioxidant capacity (TAC) of garlic extracts estimated by the commonly used reductive assays. Comparable cytotoxic activities of OSCs and phenolics (caffeic, p-coumaric, ferulic, gallic acids, and quercetin) indicate that both classes of compounds may contribute to the cytotoxic action of garlic.

The high content of antioxidants in garlic is well documented [4][5][6]16].Garlic was found to have the highest oxygen radical absorbance capacity (ORAC) among over twenty popular vegetables, which evidences high activity of its components for scavenging peroxyl radicals [17].
Garlic is also an anticancer agent.The raw garlic extract was reported to hamper the growth of various malignant cells, showing the highest efficacy among 34 raw vegetable extracts, and exerting no discernible impact on nonmalignant cells [18].Our previous study identified garlic extracts as the most cytotoxic to human ovarian cancer cells among extracts of 17 popular vegetables (including onion), fruits, and herb infusions [19].Furthermore, garlic was also reported to have anticarcinogenic properties.Consistent consumption of garlic was evidenced to reduce the risk of incidence of cancer of the lung, breast, stomach, colon, and prostate [20].
Allicin hampered the growth of murine and human malignant cells and induced their apoptosis [29][30][31].This compound prevented the induction of gastric cancer as well [32].Apart from allicin, other OSCs including ajoene, DAS, DADS, DATS, DATeS, DPDS, SAC, and SAMC, were demonstrated to decrease the proliferation of various malignant cells, as reviewed in [13,33].
SAMC, administered intragastrically, inhibited the development of xenograft tumors formed by human HO8910 and SKOV-3 cells.SAC and SAMC inhibited the migration of SKOV-3 cells and their invasiveness [34].Prolonged treatment with SAC inhibited proliferation and provoked apoptosis of A2780 human ovarian carcinoma cells and attenuated migration and invasiveness of these cells [35].
While it is generally assumed that OSCs condition the cytotoxicity of garlic to malignant cells, the determinants of the antioxidant capacity of garlic are less understood.When comparing the composition and TAC of various garlic cultivars, significant correlations were reported between the level of alliin and antioxidant capacity determined by the ABTS • reduction, DPPH • reduction, and FRAP methods [36]; nevertheless the comparison included only four cultivars, and the alliin content could be associated with the content of other compounds.Allicin was reported to act as a chain-breaking antioxidant [37], but its reaction rate constant with cumene peroxyl radicals hydroperoxide was rather low (2.6 × 10 3 M −1 s −1 ) [38].DADS was found to be an efficient lipid peroxidation terminator; nonetheless, alliin, allicin, and SAC were ineffective in the prevention of induced microsomal lipid peroxidation [39].
Garlic contains many other biologically active components beyond OSCs as well; among them are flavonoids and other phenolic compounds, steroids, saponins, polysaccharides, vitamins, and proteins, including lectins and enzymes.These constituents are likely to exhibit additive, synergistic, or antagonistic effects with OSCs [40][41][42].
Phenolics are known to be good antioxidants [43,44], but also anticancer agents [45,46], so they may be expected to have the cytotoxic effects of garlic.This study aimed to address this problem by examining the antioxidant activities of individual compounds belonging to the groups of OSCs and phenolics and their cytotoxic activities toward two ovarian cancer cell lines and human fibroblasts.

Results
The structures of the OSCs and phenolics used in this study are shown in Figures 1 and 2, respectively.
A comparison of antioxidant properties of various garlic components demonstrated negligible antioxidant activities of OSCs studied in all antioxidant assays employed.In contrast, the studied phenolics showed considerable antioxidant activities (Table 1).A comparison of antioxidant properties of various garlic components demonstrated negligible antioxidant activities of OSCs studied in all antioxidant assays employed.In contrast, the studied phenolics showed considerable antioxidant activities (Table 1).A comparison of antioxidant properties of various garlic components demonstrated negligible antioxidant activities of OSCs studied in all antioxidant assays employed.In contrast, the studied phenolics showed considerable antioxidant activities (Table 1).In the cytotoxicity experiments, two human ovarian cancer cell lines (SKOV-3 and PEO1) and a normal human fibroblast MRC-5 cell line were used.The proliferation of ovarian cancer cells and fibroblasts was inhibited by compounds belonging to both groups.Somewhat surprisingly, the cytotoxic activities of OSCs were far from striking.Allin and S-allyl-D-cysteine did not significantly decrease the viability of PEO1, SKOV-3, and MRC-5 cells at concentrations between 500 and 1500 µM.Allicin decreased the viability of MRC-5 fibroblasts at a concentration of 100 µM; nevertheless, it did not significantly lower the viability of SKOV-3 and PEO1 ovarian cancer cells in the concentration range studied.Allyl sulfide did not decrease the viability of any tested cells at concentrations up to 100 µM, but at concentrations of 200 to 500 µM, it lowered the viability of SKOV-3 and MRC-5 cells.DADS significantly decreased the survival of MRC-5 cells only at a concentration of 300 µM.DATS did not change the viability of normal MRC-5 cells in the concentration range of 0 to 50 µM, but lowered the viability of SKOV-3 and PEO1 cells at the concentration of 50 µM (Figure 3).Among the phenolic compounds present in garlic, coumaric and ferulic acids did not cause any significant change in the viability of MRC-5, SKOV-3, or PEO1 cells, within the concentration range of 0-1000 µ M. Caffeic acid decreased the viability of MRC-5 cells at a concentration of 1000 µ M and of PEO1 ovarian cancer cells in the concentration range of 300-1000 µ M. SKOV-3 cells were resistant to the effects of caffeic acid within the studied concentration range up to 1000 µ M.Among the tested compounds, gallic acid exhibited Among the phenolic compounds present in garlic, coumaric and ferulic acids did not cause any significant change in the viability of MRC-5, SKOV-3, or PEO1 cells, within the concentration range of 0-1000 µM.Caffeic acid decreased the viability of MRC-5 cells at a concentration of 1000 µM and of PEO1 ovarian cancer cells in the concentration range of 300-1000 µM.SKOV-3 cells were resistant to the effects of caffeic acid within the studied concentration range up to 1000 µM.Among the tested compounds, gallic acid exhibited the highest toxicity at concentrations of 200-1000 µM, reducing the viability of all cell lines studied.Quercetin did not decrease the viability of MRC-5 cells at concentrations up to 200 µM; nonetheless, it lowered the viability of SKOV-3 and PEO1 cells at concentrations of 75-200 µM (Figure 4).The concentrations of organosulfur compounds and phenolics lowering cell viability by 50% (IC 50 values) are shown in Table 2.  2.

Discussion
The presented results indicate the lack of reactivity of OSCs in standard antioxidant activity assays such as ABTS • and DDPH • decolorization assays and FRAP, based on the reduction in stable free radicals or Fe 3+ ions.This finding is in line with the report of Mahmutovic et al. [47] on the lack of dependence between the TAC of bulbs and leaves of various samples of garlic and ramson, as well as their OSC content.Analysis of our previous results [48] points to a lack of significant correlations between the sum of organosulfur compounds and TAC estimated by ABTS • and DDPH • decolorization and FRAP assays (r of 0.22, 0.06, and −0.39, respectively; n = 6).In contrast, the phenolic compounds studied showed considerable antioxidant activity, indicating that compounds of this class contribute to the TAC of garlic extracts and, together with ascorbic acid, can be the main determinants of TAC measured by the reductive assays.Other studies reported TAC of garlic extracts to be well correlated with the total phenolic content [48][49][50].High amounts of ascorbic acid were also found in garlic (0.16 to 0.34 mg/g in five Italian endemic varieties) [51].
Surprisingly, the cytotoxic effects of the studied compounds present in garlic, especially OSCs, found in this study were rather modest.The low cytotoxicities of OSCs in our study were partly due to our measuring the cytotoxic activity after 24 h incubation.Stronger effects are usually observed after 48 h or 72 h exposure, due to the trivial fact of proliferation of control cells, with which the amounts of treated cells are compared, during prolonged incubation.Nevertheless, much stronger cytotoxic activities were found by some authors, although the results of various studies vary markedly.The data cited below concern 24 h exposure unless stated otherwise; the values are often approximate and read from figures.Treatment with 50 nM allicin decreased human cervical squamous carcinoma SiHa cell viability to ca. 60% [52].Survival of human gastric carcinoma AGS cells was diminished to ca. 50% by 10 µg/mL, i.e., about 62 µM allicin [53].Allicin concentrations lowering the survival of MDA-MB-231 and MCF-7 breast cancer cells by 50%, corresponding to about 18 and about 7 µg/mL (about 111 and 43 µM), respectively [54].The viability of SKOV-3 cells was reduced to 50% by allicin concentration of 70-80 µg/mL (0.43-0.49mM) [55], of human U251 glioma cells by ca.40 µg/mL allicin (247 µM) [18], and of U87MG human glioblastoma cells by ca.90/mL µg (0.55 mM) allicin [56] (data for 24 h exposure).Only a ca.25% decrease in the survival of SGC7901 human gastric adenocarcinoma cells by 120 µg/mL (i.e., ca.0.74 mM) allicin was reported [57].Allicin lowered the viability of breast cancer MCF-7 and HCC-70 cells to 50% at concentrations of ca. 15 µM and ca. 10 µM, respectively, while their viability was decreased down to ca. 75% and ca.85%, respectively, by 1 mM alliin [58].However, in our comparison of the cytotoxic properties of various garlic extracts [48], the IC 50 values for decreasing cell viability correlated negatively with the sum of contents of organosulfur compounds determined, but the correlations coefficients were below the level of statistical significance (r of −0.44, −0.50, and −0.44 for SCOV-3, PEO1, and MRC-5 cells, respectively; n = 6).These results can suggest that also other components of garlic extracts contribute to their cytotoxicity.
The viability of human cervical epidermoid carcinoma Ca Ski cells was decreased to about 50% by ca. 25 µM DADS [59] and of breast cancer MCF-7 cells by 400 µM DADS [60].Diallyl disulfide reduced the survival of prostatic carcinoma LNCaP cells by about 30% by 100 µM DADS [61] and the survival of lung cancer A549 cells by about 20% by 200 µM DADS [62].The survival of colorectal cancer HCT-116 cells was decreased to ca. 80% by 400 µM DADS [63].DADS at the concentration of 1.5 mM was reported to reduce the viability of MDA-MB-468 cells to about 65% and of human lung epithelial BEAS-2B cells to about 75% [64].
There may be another important factor contributing to the variability of results concerning the cytotoxicity of garlic OSCs.These compounds are reactive, especially with thiols, forming covalent S-allyl conjugates [65][66][67].If OSCs are introduced to the medium before the addition of the medium to the cells, they may react with components of the medium, especially with proteins of the fetal serum, with the fraction of unreacted compound decreasing as a function of time before addition to the cells and penetration of the plasma membrane of the cells.Such detail as the exact time interval between the dilution of an OSC with the medium and contact of the medium with the cell is not reported in standard protocols and may differ between experimenters.Importantly, the lower cytotoxicity values may be more relevant since, in the body, OSCs released from garlic may have even more opportunities to react with body components before reaching target cells.
The phenolic compounds present in garlic have also significant cytotoxic properties.p-Coumaric acid decreased the proliferation of neuroblastoma N2a cells by 50% at the concentration of 104 µM after 24 h [68], of A375 and B16 melanocytes at the concentrations of 2.5 and 2.8 mM, respectively [69], and of colon carcinoma HT-29 cells and human colorectal carcinoma HCT-15 cells with at the concentrations of 1.4 and 1.6 mM, respectively, after 48 h exposure [70].
Caffeic acid decreased the viability of HT-1080 fibrosarcoma cells at the concentration of 30 µM [74], of MDA-MB-231 cells at the concentration of 151 µM [75], and of human cervical cancer HeLa cells by 50% at the concentration of about 2 mM (after 24 h exposure) [76].
The values reported in the literature and found in this study needed to decrease the viability of cancer cells are mostly too high to be obtained in vivo; moreover, colonic fermentation decreasing their content in the food and limiting intestinal absorption as well as metabolism, may decrease their bioavailability.It was found, e.g., that colonic fermentation of garlic results in a 44 and 41% loss of phenolics in fresh garlic and black garlic, respectively, and a 33% decrease in OSCs [86].Nevertheless, higher concentrations of these compounds can be released from food products and encountered by cells of the digestive tract or during a topical treatment.
Comparable sensitivity of cancer cells to garlic OSCs and phenolics found in our study suggests that not only OSCs but both types of compounds may contribute to the anticancer activity of garlic.The efficiency of different types of garlic in preventing fibrosarcoma growth in mice was found to correlate with the contents of not only allicin but also phenolics and flavonoids [87].The antiproliferative activity of extracts of several varieties of Italian garlic did not correlate with their content of OSCs, and it was supposed that the cytotoxicity of garlic can be ascribed to the synergistic action of many metabolites, including ascorbate [51].The results presented in this study seem to support this view.Organosulfur compounds may be more important as they are present in higher amounts than phenolics, but the latter can also have a contribution to the cytotoxicity of garlic for cancer cells.Moreover, interactions between various garlic constituents may affect the cytotoxic effects of garlic extracts.This question deserves further studies.
Stock solutions of allicin, AS, DADS, DATS, caffeic, coumaric, and ferulic and gallic acids, as well as quercetin were prepared in DMSO.The effects of the solvent (if any) were subtracted from the effects exerted by the solutions containing DMSO (in all cases at final not exceeding 0.2%).Stock solutions of alliin and S-allyl-D-cysteine were made in PBS and diluted with the cell culture media.

ABTS • Reduction Assay
A modification [88] of the ABTS • decolorization assay [89] was employed.Briefly, various volumes of solutions of the studied compounds were introduced to wells of a 96-well microplate, each prefilled 200 µL of ABTS • solution.The stock ABTS • solution was diluted with PBS so that 200 µL of the sample had an initial absorbance of 1.0 at 734 nm in a well of a 96-well microplate.The drop in absorbance after 30 min incubation at room temperature was read as a measure of the antioxidant activity.

DPPH • Reduction Assay
The assay was performed as described in a previous article [90].Briefly, increasing volumes of solutions of the studied compounds were introduced to wells of a 96-well microplate, each with 200 µL of 0.3 mM DPPH • solution in methanol.The reaction was allowed to proceed for 30 min at ambient temperature in the dark, and absorbance at 517 nm was read.The absorbance decrease was a measure of the antioxidant activity.

The Ferric Reducing Antioxidant Power (FRAP) Assay
The method proposed by Benzie and Strain [91] was employed with a slight modification.Briefly, increasing volumes of solutions of the studied compounds were added to wells of a 96-well microplate prefilled with 200 µL of the working solution composed of 0.3 M acetate buffer, pH 3.6 (10 volumes), 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), in 40 mM HCl (1 volume) and 20 mM FeCl 3 (1 volume), was prepared immediately before use.After 30 min incubation at ambient temperature and the absorbance of the Fe 2+ -TPTZ complex was read at 593 nm.

Calculation of the Antioxidant Activity
All assays were standardized with respect to Trolox.The antioxidant activity was calculated in each case as a ratio of the slope of the dependence of absorbance change on the concentration of a studied compound to the slope of the dependence of absorbance change of standard samples concerning Trolox on the concentration of Trolox.Antioxidant activities were expressed in moles of Trolox equivalents (TE)/mol of a studied compound, as described previously [88].

Cell Culture
The PEO1 cells, SKOV-3 cells, and MRC-5 cells were grown at 37 • C and 5% CO 2 in RPMI + GlutaMAX medium, McCoy's 5A medium, and the DMEM + GlutaMAX medium, respectively.The media were supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin.The cell viability was evaluated using the Trypan Blue exclusion test.Cells were counted in a Thoma hemocytometer (Superior Marienfeld, Lauda-Königshofen, Germany).
In the cytotoxicity experiments, cells were grown in wells of sterile 96-well plates.The seeding density was 1 × 10 4 for SKOV-3 and MRC-5 cells and 1.5 × 10 4 for PEO1 cells.The cells were allowed to grow in the incubator for 24 h.Then, the medium was removed and new medium with an examined compound was added.The stock solutions of the compounds studied diluted with the appropriate cell medium were filtered before the addition to the cells using 0.22 µm filters.No more than 0.02% of DMSO was present in the media, and it had no discernible impact on the treated cells.Cells not exposed to any compound served as a control.After another 24 h incubation, the medium was gently aspirated, and the wells were added with 2% sterile Neutral Red solution (100 µL).The plates were then incubated (37 • C, 5% CO 2 ) for one hour.The Neutral Red solution was aspirated, and the wells were washed two times with warm (37 • C) PBS.Subsequently, 100 µL of a permeabilization mixture (water/ethanol/glacial acetic acid, 50/49/1, v/v/v) was introduced to each well.The plates were shaken at 700 rpm for 20 min and absorbance was read at a wavelength of 540 nm against 620 nm.

Statistical Analysis
To assess the variances of differences between control cells and cells treated with appropriate compounds, the Kruskal-Wallis test was conducted (n ≥ 6 biological replicates), assuming differences with p ≤ 0.05 as statistically significant.The statistical analysis was carried out using the STATISTICA software package (version 13.1, StatSoft Inc., 2016, Tulsa, OK, USA).

Conclusions
Garlic organosulfur compounds show negligible activity in the common reductive assays of antioxidant activity (ABTS • and DPPH • decolorization assays and FRAP), so they cannot contribute significantly to the antioxidant capacity of garlic extracts measured by these assays.Apart from OSCs, phenolics inhibit cell proliferation and can contribute to the cytotoxicity of garlic.

Figure 1 .
Figure 1.Structures of organosulfur compounds of garlic used in this study.

Figure 2 .
Figure 2. Structures of phenolic compounds of garlic used in this study.

Figure 1 .
Figure 1.Structures of organosulfur compounds of garlic used in this study.

Figure 1 .
Figure 1.Structures of organosulfur compounds of garlic used in this study.

Figure 2 .
Figure 2. Structures of phenolic compounds of garlic used in this study.

Figure 2 .
Figure 2. Structures of phenolic compounds of garlic used in this study.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 200 µ M; nonetheless, it lowered the viability of SKOV-3 and PEO1 cells at concentration of 75-200 µ M (Figure 4).The concentrations of organosulfur compounds and phenoli lowering cell viability by 50% (IC50 values) are shown in Table
Note: nd, determination of IC 50 not possible on the basis of obtained data.